Part:BBa_K3888005:Design

UppE Upstream Flanking Region

Figure 1: Illustration of the general form of plasmid used to create marked knockout strains of R. palustris.

The origin of replication (ori), origin of transfer (oriT) and sacB (blue) are part of the delivery plasmid. The green sequence is inserted into the multiple cloning site of the delivery plasmid. The left and right fragments (green) are sequences amplified from the R. palustris genome and used in overlap extension PCR to generate a recombinant product containing (a deletion and a restriction site）. The restriction site is used to introduce the resistance cassette (purple). This interrupts the gene even if no deletion is present, and allows selection of successful transformants.
According to this model, we constructed UppE knockout plasmid below.

Figure 2: Deletion plasmid of UppE constructed according to the method mentioned above. UppE Upstream Flanking region is located from 1021bp to 2056bp.

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 694
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 694
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 694
  Illegal BglII site found at 333
  Illegal XhoI site found at 826
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 694
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 694
  Illegal NgoMIV site found at 38
  Illegal NgoMIV site found at 119
  Illegal NgoMIV site found at 157
  Illegal NgoMIV site found at 831
  Illegal NgoMIV site found at 943
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 538
  

Design Notes

Flanking region for 1k bp

Source

Rhodopseudomonas palustris CGA009

References

1. Fritts, Ryan K., et al. "A Rhizobiales-specific unipolar polysaccharide adhesin contributes to Rhodopseudomonas palustris biofilm formation across diverse photoheterotrophic conditions."Applied and environmental microbiology 83.4 (2017): e03035-16